(F-222) Analyzing the Targeting and Stimuli-Responsive Components of a Multi-Functional Peptide to Induce Gene Knockdown in Glioblastoma Cells in vitro.

RNA interference (RNAi) is a naturally occurring biological process in which sequence-specific short-interfering RNA (siRNA) induces gene silencing through mRNA degradation. Because of its high specificity, siRNA has recently been used to target oncogenes as a cancer therapeutic. However, siRNA has multiple delivery barriers due to its size and negative charge, requiring a carrier for successful cellular delivery. The ideal carrier for siRNA protects it from enzymatic degradation, allows for cellular internalization, and promotes endosomal escape of the siRNA into the cytosol, where it is bioactive. Peptides are a promising delivery vehicle for siRNA because of their functional and structural diversity. This study focuses on a targeting peptide, a stimuli-responsive peptide, and a novel tandem peptide comprising both the targeting and stimuli-responsive peptide components. The targeting peptide, GE11, targets the epidermal growth factor receptor (EGFR), which is overexpressed in many cancers. The stimuli-responsive peptide, 599, is activated in an acidic environment, such as an endosome, where it undergoes a conformational change that enables endosome disruption and release of cargo into the cytosol. Finally, the novel tandem peptide, GE11-599, combines these peptides to achieve both functions. This study aims to analyze the tandem, targeting, and stimuli-responsive peptides and their ability to deliver bioactive siRNA into cancer cells to induce gene silencing.

The three peptides, GE11, 599, and GEll-599, were custom synthesized and purified by Lifetein LLC. Human glioblastoma cell lines, U118MG and U87MG, and human embryonic kidney cell line HEK293-GFP were obtained from American Type Culture Collection (ATCC). Peptide/siRNA complexes were formed by incubation of the GE11 peptide or 599 peptide with Cy5-labeled siRNA at increasing N:P molar ratios for 20 minutes to allow self-assembly. Peptide/siRNA complexes were incubated with GBM and HEK 293 cells for 4 hours to allow cellular uptake followed by imaging Fluorescence microscopy analysis was conducted to evaluate the internalization of siRNA into U118 and U87 cells. EEA1 was used for immunofluorescent imaging of endosomes to determine the ability of the stimuli-responsive peptides to mediate endosomal disruption. A co-culture assay using U87 and HEK293-GFP cells was performed to evaluate the ability of the targeting peptides to target EGFR-expressing cells. A western blot was completed with these cell lines to confirm basal EGFR expression. qPCR was conducted to analyze the ability of the peptide-siRNA complexes to silence a target oncogene.

Uptake imaging in the U118 and U87 cell lines indicated successful internalization of siRNA into both cell lines using the GE11 or 599 peptides. siRNA is efficiently being delivered to the cell using GE11 and GE11-599 in U87 cells (Fig 1A). To analyze the ability of the GE11 peptide and GE11-599 tandem peptide to target EGFR, a co-culture assay was conducted and revealed higher internalization of siRNA into the EGFR+ U87 cells than the healthy HEK293-GFP cells. This data suggests the targeting and tandem peptides deliver siRNA specifically into cells expressing EGFR. A western blot was used to evaluate the basal expression of EGFR in these cell lines, showing EGFR expression in both cell lines, but a higher expression in the U87 cells. To investigate the functionality of the stimuli-responsive peptide, endosomal escape images showed that at most N:P ratios of the stimuli-responsive 599 peptide and the GE11-599 tandem peptide, the siRNA was not colocalized within the endosome (Fig 1B). This result confirmed the functionality of the tandem peptide for disrupting the endosome to release siRNA into the cytosol, where it can be active. Furthermore, qPCR analyzed the bioactivity of the delivered siRNA, demonstrating efficient knockdown of a target oncogene.


In conclusion, the results demonstrate the functionality of the GE11 targeting peptide to target EGFR and the stimuli-responsive 599 peptide to disrupt the endosome. The novel tandem peptide can perform both the targeting and stimuli-responsive functions and efficiently silence oncogenes, making it a promising cancer therapeutic delivery system. Future studies include an EGFR inhibition study to further analyze the internalization mechanism, western blotting to determine gene silencing on the protein level via, and downstream analysis of the effect of silencing specific oncogenes. 

Acknowledgments: Funding provided by the National Science Foundation EPSCoR Award #OIA-1655740 and NSF Career Award #2046694